Fig. 5: Vps15 transcriptionally coactivates Bmal1.
From: Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis
a, Left: luciferase assay in HEK293T cells co-transfected with the E-box-Luc reporter and EV or BMAL1–CLOCK with or without VPS15WT, VPS15Mut1, CBP or p300. Relative luminescence presented as fold difference compared with cells transfected with E-box-Luc + EV. Data are the mean ± s.e.m. (independent experiments, n = 4 for BMAL1–CLOCK + VPS15Mut1/CBP, n = 5 for BMAL1–CLOCK + p300, n = 8 for BMAL1–CLOCK + EV and n = 12 for BMAL1–CLOCK + VPS15WT). Right: representative immunoblot with anti-Flag showing the expression levels of BMAL1, CLOCK, VPS15 and GAPDH as the loading control (right). b, Left: luciferase assay in HEK293T cells co-transfected with E-box-Luc reporter construct and EV or plasmids expressing BMAL1 and CLOCK with or without Cry1, VPS15WT or VPS15Mut1. Relative luminescence is presented as the fold difference compared with E-box-Luc-transfected cells. Data are the mean ± s.e.m. (n = 4 independent experiments). Right: representative immunoblot showing the expression levels of Cry1, BMAL1, CLOCK, VPS15 and GAPDH as the loading control. c, ChIP–qPCR analyses of Bmal1 and Vps15 enrichment at the promoters of the indicated genes in the liver tissue of 5-week-old male mice (ZT6). Data are the mean ± s.e.m. fold enrichment (n = 6 mice). a–c, *P < 0.05; two-tailed unpaired Student’s t-test. d, Vps15 binding peak profile and heatmap for the livers (n = 2 mice) of WT male mice (ZT6; 5 weeks old). Peaks are ordered by their signal strength and each row shows the promoter region from −3 kb to +3 kb from the TSS. e, HOMER motif analysis of Vps15 peaks. Identified consensus motifs are shown with their respective significance calculated with HOMER and the percentage of target coverage in all ChIP peaks. f, Analysis of GO Biological process using enrichGO showing significantly enriched genes for which chromatin binding of Vps15 was detected in WT liver and for which Bmal1 chromatin enrichment and transcript levels were downregulated in the livers of Vps15LKO mice. Source data and unprocessed blots are provided.
